Proteinase inhibitor, precursor thereof and genetic sequences encoding same

ABSTRACT

The present invention relates generally to proteinase inhibitors, a precursor thereof and to genetic sequences encoding same. More particularly, the present invention relates to a nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes a type II serine proteinase inhibitor (PI) precursor from a plant wherein said precursor comprises at least three PI monomers and wherein at least one of said monomers has a chymotrypsin specific site and at least one other of said monomers has a trypsin specific site.

The present invention relates generally to proteinase inhibitors, aprecursor thereof and to genetic sequences encoding same.

Nucleotide and amino acid sequences are referred to herein by sequenceidentity numbers (SEQ ID NOs) which are defined after the bibliography.A general summary of the SEQ ID NOs is provided before the examples.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word "comprise", or variations such as"comprises" or "comprising", will be understood to imply the inclusionof a stated element or integer or group of elements or integers but notthe exclusion of any other element or integer or group of elements orintegers.

Several members of the families Solanaceae and Fabaceae accumulateserine proteinase inhibitors in their storage organs and in leaves inresponse to wounding (Brown and Ryan, 1984; Richardson, 1977). Theinhibitory activities of these proteins are directed against a widerange of proteinases of microbial and animal origin, but rarely againstplant proteinases (Richardson, 1977). It is believed that theseinhibitors are involved in protection of the plants against pathogensand predators. In potato tubers and legume seeds, the inhibitors cancomprise 10% or more of the stored proteins (Richardson, 1977), while inleaves of tomato and potato (Green and Ryan, 1972), and alfalfa (Brownand Ryan, 1984), proteinase inhibitors can accumulate to levels of 2% ofthe soluble protein within 48 hours of insect attack, or other types ofwounding (Brown & Ryan, 1984; Graham et al., 1986). High levels of theseinhibitors (up to 50% of total soluble protein) are also present inunripe fruits of the wild tomato, Lycopersicon peruvianum (Pearce etal., 1988).

There are two families of serine proteinase inhibitors in tomato andpotato (Ryan, 1984). Type I inhibitors are small proteins (monomer Mr8100) which inhibit chymotrypsin at a single reactive site (Melville andRyan, 1970; Plunkett et al., 1982). Inhibitors of the type II familygenerally contain two reactive sites, one of which inhibits chymotrypsinand the other trypsin (Bryant et al., 1976; Plunkett et al., 1982). Thetype II inhibitors have a monomer Mr of 12,300 (Plunkett et al., 1982).Proteinase inhibitor I accumulates in etiolated tobacco (Nicotianatabacum) leaves (Kuo et al., 1984), and elicitors from Phytophthoraparasitica var. nicotianae were found to induce proteinase inhibitor Iaccumulation in tobacco cell suspension cultures (Rickauer et al.,1989).

There is a need to identify other proteinase inhibitors and toinvestigate their potential use in the development of transgenic plantswith enhanced protection against pathogens and predators. In accordancewith the present invention, genetic sequences encoding a proteinaseinhibitor precursor have been cloned. The precursor has multi-proteinaseinhibitor domains and will be useful in developing a range of transgenicplants with enhanced proteinase inhibitor expression. Such plants willhave enhanced protective properties against pathogens and predators. Thegenetic constructs of the present invention will also be useful indeveloping vaccines for ingestion by insects which are themselvespredators or which act as hosts for plant pathogens. The recombinantprecursor or monomeric inhibitors will also be useful in topical spraysand in assisting animals in feed digestion.

Accordingly, one aspect of the present invention relates to a nucleicacid molecule comprising a sequence of nucleotides which encodes or iscomplementary to a sequence which encodes a type II serine proteinaseinhibitor (PI) precursor from a plant wherein said precursor comprisesat least three PI monomers and wherein at least one of said monomers hasa chymotrypsin specific site and at least one other of said monomers hasa trypsin specific site.

The "nucleic acid molecule" of the present invention may be RNA or DNA(eg cDNA), single or double stranded and linear or covalently closed.The nucleic acid molecule may also be genomic DNA corresponding to theentire gene or a substantial portion thereof or to fragments orderivatives thereof. The nucleotide sequence may correspond to thenaturally occurring nucleotide sequence of the genomic or cDNA clone ormay contain single or multiple nucleotide substitutions, deletionsand/or additions thereto. All such variants in the nucleic acid moleculeeither retain the ability to encode at least one monomer or active partthereof or are useful as hybridisation probes or polymerase chainreaction (PCR) primers for the same or similar genetic sequences inother sources.

Preferably, the PI precursor comprises at least four, more preferably atleast five and even more preferably at least six PI monomers. Still morepreferably, the PI precursor further comprises a signal sequence. The PIprecursor of the present invention preferably comprises amino acidsequences which are process sites for cleavage into individual monomers.

The term "precursor" as used herein is not intended to place anylimitation on the utility of the precursor molecule itself or arequirement that the molecule first be processed into monomers before PIactivity is expressed. The precursor molecule has PI activity and thepresent invention is directed to the precursor and to the individualmonomers of the precursor.

Furthermore, the present invention extends to a nucleic acid moleculecomprising a sequence of nucleotides which encodes or is complementaryto a sequence which encodes a hybrid type II serine PI precursor whereinsaid precursor comprises at least two monomers from different PIs. Theat least two monomers may be modified such as being unable to beprocessed into individual monomers or may retain the ability to be soprocessed. Preferably, at least one of said monomers has a chymotrypsinspecific site and the other of said monomers has a trypsin specificsite. Preferably there are at least three monomers, more preferably atleast four monomers, still more preferably at least five monomers andyet still more preferably at least six monomers wherein at least two arefrom different PIs. In a most preferred embodiment, at least one of saidmonomers is a thionin. Such hybrid PI precursors and/or monomers thereofare particularly useful in generating molecules which are "multi-valent"in that they are active against a range of pathogens and predators suchas both fungi and insects. Accordingly, reference herein to "PIprecursor" includes reference to hybrid molecules.

The present invention is exemplified by the isolation of the subjectnucleic acid molecule from Nicotiana alata which has the followingnucleotide sequence (SEQ ID NO. 1) and a corresponding amino acidsequence (SEQ ID NO. 3):

                                            AAG GCT TGT ACC TTA AAC                                                         Lys Ala Cys Thr Leu Asn                                                      - TGT GAT CCA AGA ATT GCC TAT                                               GGA GTT TGC CCG CGT TCA GAA GAA                                               AAG                                      Cys Asp Pro Arg Ile Ala Tyr Gly Val Cys Pro Arg Ser Glu Glu Lys                                                      - AAG AAT GAT CGG ATA TGC ACC                                               AAC TGT TGC GCA GGC ACG AAG GGT                                               TGT                                      Lys Asn Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Thr Lys Gly Cys                                                      - AAG TAC TTC AGT GAT GAT GGA                                               ACT TTT GTT TGT GAA GGA GAG TCT                                               GAT                                      Lys Tyr Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Cly Glu Ser Asp                                                      - CCT AGA AAT CCA AAG GCT TGT                                               ACC TTA AAC TGT GAT CCA AGA ATT                                               GCC                                      Pro Arg Asn Pro Lys Ala Cys Thr Leu Asn Cys Asp Pro Arg Ile Ala                                                      - TAT GGA GTT TGC CCG CGT TCA                                               GAA GAA AAG AAG AAT GAT CGG ATA                                               TGC                                      Tyr Gly Val Cys Pro Arg Ser Glu Glu Lys Lys Asn Asp Arg Ile Cys                                                      - ACC AAC TGT TGC GCA GGC ACG                                               AAG GGT TGT AAG TAC TTC AGT GAT                                               GAT                                      Thr Asn Cys Cys Ala Gly Thr Lys Gly Cys Lys Tyr Phe Ser Asp Asp                                                      - GGA ACT TTT GTT TGT GAA GGA                                               GAG TCT GAT CCT AGA AAT CCA AAG                                               GCT                                      Gly Thr Phe Val Cys Glu Gly Glu Ser Asp Pro Arg Asn Pro Lys Ala                                                      - TGT CCT CGG AAT TGC GAT CCA                                               AGA ATT GCC TAT GGG ATT TGC CCA                                               CTT                                      Cys Pro Arg Asn Cys Asp Pro Arg Ile Ala Tyr Gly Ile Cys Pro Leu                                                      - GCA GAA GAA AAG AAG AAT GAT                                               CGG ATA TGC ACC AAC TGT TGC GCA                                               GGC                                      Ala Glu Glu Lys Lys Asn Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly                                                      - AAA AAG GGT TGT AAG TAC TTT                                               AGT GAT GAT GGA ACT TTT GTT TGT                                               GAA                                      Lys Lys Gly Cys Lys Tyr Phe Ser Asp Asp Gly Thr Phe Val Cys Glu                                                      - GGA GAG TCT GAT CCT AAA AAT                                               CCA AAG GCC TGT CCT CGG AAT TGT                                               GAT                                      Gly Glu Ser Asp Pro Lys Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp                                                      - GGA AGA ATT GCC TAT GGG ATT                                               TGC CCA CTT TCA GAA GAA AAG AAG                                               AAT                                      Gly Arg Ile Ala Tyr Gly Ile Cys Pro Leu Ser Glu Glu Lys Lys Asn                                                      - GAT CGG ATA TGC ACC AAC TGC                                               TGC GCA GGC AAA AAG GGT TGT AAG                                               TAC                                      Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Lys Lys Gly Cys Lys Tyr                                                      - TTT AGT GAT GAT GGA ACT TTT                                               GTT TGT GAA GGA GAG TCT GAT CCT                                               AAA                                      Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gly Glu Ser Asp Pro Lys                                                      - AAT CCA AAG GCT TGT CCT CGG                                               AAT TGT GAT GGA AGA ATT GCC TAT                                               GGG                                      Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp Gly Arg Ile Ala Tyr Gly                                                      - ATT TGC CCA CTT TCA GAA GAA                                               AAG AAG AAT GAT CGG ATA TGC ACA                                               AAC                                      Ile Cys Pro Leu Ser Glu Glu Lys Lys Asn Asp Arg Ile Cys Thr Asn                                                      - TGT TGC GCA GGC AAA AAG GGC                                               TGT AAG TAC TTT AGT GAT GAT GGA                                               ACT                                      Cys Cys Ala Gly Lys Lys Gly Cys Lys Tyr Phe Ser Asp Asp Gly Thr                                                      - TTT GTT TGT GAA GGA GAG TCT                                               GAT CCT AGA AAT CCA AAC GCC TGT                                               CCT                                      Phe Val Cys Glu Gly Glu Ser Asp Pro Arg Asn Pro Lys Ala Cys Pro                                                      - CCG AAT TGT GAT GGA AGA ATT                                               GCC TAT GGA ATT TGC CCA CTT TCA                                               GAA                                      Arg Asn Cys Asp Gly Arg Ile Ala Tyr Gly Ile Cys Pro Leu Ser Glu                                                      - GAA AAG AAG AAT GAT CGG ATA                                               TGC ACC AAT TGT TGC GCA GGC AAG                                               AAG                                      Glu Lys Lys Asn Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Lys Lys                                                      - GGC TGT AAG TAC TTT AGT GAT                                               GAT GGA ACT TTT ATT TGT GAA GGA                                               GAA                                      Gly Cys Lys Tyr Phe Ser Asp Asp Gly Thr Phe Ile Cys Glu Gly Glu                                                      - TCT GAA TAT GCC AGC AAA GTG                                               GAT GAA TAT GTT GGT GAA GTG GAG                                               AAT                                      Ser Glu Tyr Ala Ser Lys Val Asp Glu Tyr Val Gly Glu Val Glu Asn                                                      - GAT CTC CAG AAG TCT AAG GTT                                               GCT GTT TCC                              Asp Leu Gln Lys Ser Lys Val Ala Val Ser                                 

This is done, however, with the understanding that the present inventionextends to an equivalent or substantially similar nucleic acid moleculefrom any other plant. By "equivalent" and "substantially similar" ismeant at the level of nucleotide sequence, amino acid sequence, antibodyreactivity, monomer composition and/or processing of the precursor toproduce monomers. For example, a nucleotide sequence having a percentagesequence similarity of at least 55%, such as about 60-65%, 70-75%,80-85% and over 90% when compared to the sequence of SEQ ID NO. 1 wouldbe considered "substantially similar" to the subject nucleic acidmolecule provided that such a substantially similar sequence encodes aPI precursor having at least three monomers and preferably four, five orsix monomers as hereinbefore described.

In a particularly preferred embodiment, the nucleic acid moleculefurther encodes a signal sequence 5' to the open reading frame and/or anucleotide sequence 3' of the coding region providing a full nucleotidesequence as follows (SEQ ID NO. 2):

    CGAGTAAGTA TGGCTGTTCA CAGAGTTAGT TTCCTTGCTC TCCTCCTCTT ATTTGGAATG                - TCTCTGCTTG TAAGCAATGT GGAACATGCA GATGCC AAG GCT TGT ACC TTA AAC                                                         Lys Ala Cys Thr Leu Asn                                                  - TGT GAT CCA AGA ATT GCC TAT                                               GGA GTT TGC CCG CGT TCA GAA GAA                                               AAG                                     Cys Asp Pro Arg Ile Ala Tyr Gly Val Cys Pro Arg Ser Glu Glu Lys                                                       - AAG AAT GAT CGG ATA TGC ACC                                               AAC TGT TGC GCA GGC ACG AAG GGT                                               TGT                                     Lys Asn Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Thr Lys Gly Cys                                                       - AAG TAC TTC AGT GAT GAT GGA                                               ACT TTT GTT TGT GAA GGA GAG TCT                                               GAT                                     Lys Tyr Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gly Glu Ser Asp                                                       - CCT AGA AAT CCA AAG GCT TGT                                               ACC TTA AAC TGT GAT CCA AGA ATT                                               GCC                                     Pro Arg Asn Pro Lys Ala Cys Thr Leu Asn Cys Asp Pro Arg Ile Ala                                                       - TAT GGA GTT TGC CCG CGT TCA                                               GAA GAA AAG AAG AAT GAT CGG ATA                                               TGC                                     Tyr Gly Val Cys Pro Arg Ser Glu Glu Lys Lys Asn Asp Arg Ile Cys                                                       - ACC AAC TGT TGC GCA GGC ACG                                               AAG GGT TGT AAG TAC TTC AGT GAT                                               GAT                                     Thr Asn Cys Cys Ala Gly Thr Lys Gly Cys Lys Tyr Phe Ser Asp Asp                                                       - GGA ACT TTT GTT TGT GAA GGA                                               GAG TCT GAT CCT AGA AAT CCA AAG                                               GCT                                     Gly Thr Phe Val Cys Glu Gly Glu Ser Asp Pro Arg Asn Pro Lys Ala                                                       - TGT CCT CGG AAT TGC GAT CCA                                               AGA ATT GCC TAT GGG ATT TGC CCA                                               CTT                                     Cys Pro Arg Asn Cys Asp Pro Arg Ile Ala Tyr Gly Ile Cys Pro Leu                                                       - GCA GAA GAA AAG AAG AAT GAT                                               CGG ATA TGC ACC AAC TGT TGC GCA                                               GGC                                     Ala Glu Glu Lys Lys Asn Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly                                                       - AAA AAG GGT TGT AAG TAC TTT                                               AGT GAT GAT GGA ACT TTT GTT TGT                                               GAA                                     Lys Lys Gly Cys Lys Tyr Phe Ser Asp Asp Gly Thr Phe Val Cys Glu                                                       - GGA GAG TCT GAT CCT AAA AAT                                               CCA AAG GCC TGT CCT CGG AAT TGT                                               GAT                                     Gly Glu Ser Asp Pro Lys Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp                                                       - GGA AGA ATT GCC TAT GGG ATT                                               TGC CCA CTT TCA GAA GAA AAG AAG                                               AAT                                     Gly Arg Ile Ala Tyr Gly Ile Cys Pro Leu Ser Glu Glu Lys Lys Asn                                                       - GAT CGG ATA TGC ACC AAC TGC                                               TGC GCA GGC AAA AAG GGT TGT AAG                                               TAC                                     Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Lys Lys Gly Cys Lys Tyr                                                       - TTT AGT GAT GAT GGA ACT TTT                                               GTT TGT GAA GGA GAG TCT GAT CCT                                               AAA                                     Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gly Glu Ser Asp Pro Lys                                                       - AAT CCA AAG GCT TGT CCT CGG                                               AAT TGT GAT GGA AGA ATT GCC TAT                                               GGG                                     Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp Gly Arg Ile Ala Tyr Gly                                                       - ATT TGC CCA CTT TCA GAA GAA                                               AAG AAG AAT GAT CGG ATA TGC ACA                                               AAC                                     Ile Cys Pro Leu Ser Glu Glu Lys Lys Asn Asp Arg Ile Cys Thr Asn                                                       - TGT TGC GCA GGC AAA AAG GGC                                               TGT AAG TAC TTT AGT GAT GAT GGA                                               ACT                                     Cys Cys Ala Gly Lys Lys Gly Cys Lys Tyr Phe Ser Asp Asp Gly Thr                                                       - TTT GTT TGT GAA GGA GAG TCT                                               GAT CCT AGA AAT CCA AAG GCC TGT                                               CCT                                     Phe Val Cys Glu Gly Glu Ser Asp Pro Arg Asn Pro Lys Ala Cys Pro                                                       - CGG AAT TGT GAT GGA AGA ATT                                               GCC TAT GGA ATT TGC CCA CTT TCA                                               GAA                                     Arg Asn Cys Asp Gly Arg Ile Ala Tyr Gly Ile Cys Pro Leu Ser Glu                                                       - GAA AAG AAG AAT GAT CGG ATA                                               TGC ACC AAT TGT TGC GCA GGC AAG                                               AAG                                     Glu Lys Lys Asn Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Lys Lys                                                       - GGC TGT AAG TAC TTT AGT GAT                                               GAT GGA ACT TTT ATT TGT GAA GGA                                               GAA                                     Gly Cys Lys Tyr Phe Ser Asp Asp Gly Thr Phe Ile Cys Glu Gly Glu                                                       - TCT GAA TAT GCC AGC AAA GTG                                               GAT GAA TAT GTT GGT GAA GTG GAG                                               AAT                                     Ser Glu Tyr Ala Ser Lys Val Asp Glu Tyr Val Gly Glu Val Glu Asn                                                       - GAT CTC CAG AAG TCT AAG GTT                                               GCT GTT TCC TAAGTCCTAA CTAATAATAT       Asp Leu Gln Lys Ser Lys Val Ala Val Ser                                        - GTAGTCTATG TATGAAACAA AGGCATGCCA ATATGCTCTG TCTTGCCTGT AATCTGTAAT                                                  - ATGGTAGTGG AGCTTTTCCA                                                     CTGCCTGTTT AATAAGAAAT GGAGCACTAG                                              TTTGTTTTAG                               - TTAAAAAAAA AAAAAAAAAA                                                

including substantially similar variants thereof.

Accordingly, a preferred embodiment of the present invention provides anucleic acid molecule comprising a sequence of nucleotides as set forthin SEQ ID NO. 1 or 2 which encodes or is complementary to a sequencewhich encodes a type II serine PI precursor from Nicotiana alata orhaving at least 55% similarity to said precursor or at least one domaintherein wherein said precursor comprises a signal peptide and at leastfive monomers and wherein one of said monomers has a chymotrypsinspecific site and four of said monomers have trypsin specific sites.

In still a more preferred embodiment, the nucleic acid molecule is acDNA molecule and comprises a nucleotide sequence generally as set forthin SEQ ID NO. 1 or 2 or being substantially similar thereto ashereinbefore defined to the whole of said sequence or to a domainthereof.

Another aspect of the present invention is directed to a nucleic acidmolecule comprising a sequence of nucleotides which encodes or iscomplementary to a sequence which encodes a single type II serine PIhaving either a chymotrypsin specific site or a trypsin specific siteand wherein said PI is a monomer of a precursor PI having at least threemonomers of which at least one of said monomers has a chymotrypsin siteand the other of said monomers has a trypsin site. Preferably, however,the precursor has four, five or six monomers and is as hereinbeforedefined.

In its most preferred embodiment, the plant is N. alata (Link et Otto)having self-incompatibility genotype S₁ S₃, S₃ S₃ or S₆ S₆, and thenucleic acid molecule is isolatable from or complementary to geneticsequences isolatable from stigmas and styles of mature plants. Thecorresponding mRNA is approximately 1.4 kb and the cDNA has sixconserved domains wherein the first two domains are 100% identical andcontain chymotrypsin-specific sites (Leu-Asn). The third, fourth andfifth domains share 95-98% identity and have sites specific for trypsin(Arg-Asn). A sixth domain which also has a trypsin specific site hasless identity to the third, fourth and fifth domains (79-90%) due mainlyto a divergent 3' sequence (see Table 1). The preferred PI inhibitor ofthe present invention has a molecular weight of approximately 42-45 kDawith an approximately 29 amino acid signal sequence.

The N-terminal sequence of the monomeric PI is represented in each ofthe six repeated domains in the predicted sequence of the PI precursorprotein. Thus, it is likely that the PI precursor protein is cleaved atsix sites to produce seven peptides. Six of the seven peptides, peptides2, 3, 4, 5, 6 and 7 (FIG. 1, residues 25-82 [SEQ ID NO. 5], 83-140 [SEQID NO. 6], 141-198 [SEQ ID NO. 7], 199-256 [SEQ ID NO. 8], 257-314 [SEQID NO. 9] and 315-368 [SEQ ID NO. 9], respectively), would be in thesame molecular weight range as the monomeric PI (about 6 kDa) and wouldhave the same N-terminal sequence. Peptide 7 does not contain aconsensus site for trypsin or chymotrypsin. Peptide 1 (residues 1-24[SEQ ID NO. 4], FIG. 1) is smaller than 6 kD, has a different N-terminusand was not detected in a purified monomeric PI preparation. It could beenvisaged that peptide 1 and peptide 7 would form a functionalproteinase inhibitor with the inhibitory site on peptide 1 held in thecorrect conformation by disulphide bonds formed between the twopeptides.

Although not intending to limit the present invention to any onehypothesis, the PI precursor may be processed by a protease responsible,for example, for cleavage of an Asn-Asp linkage, to produce thebioactive monomers. More particularly, the protease sensitive sequenceis R₁ -X₁ -X₂ -Asn-Asp-R₂ where R₁, R₂, X₁ and X₂ are defined below. Thediscovery of such a sequence will enable the engineering of peptides andpolypeptides capable of being processed in a plant by cleavage of theprotease sensitive sequence. According to this aspect of the presentinvention there is provided a protease sensitive peptide comprising theamino acid sequence:

    --X.sub.1 --X.sub.2 --Asn--Asp--

wherein X₁ and X₂ are any amino acid but are preferably both Lysresidues. The protease sensitive peptide may also be represented as:

    R.sub.1 --X.sub.1 --X.sub.2 --Asn--Asp--R.sub.2

wherein X₁ and X₂ are preferably the same and are preferably both Lysresidues and wherein R₁ and R₂ are the same or different, any D or Lamino acid, a peptide, a polypeptide, a protein, or a non-amino acidmoiety or molecule such as, but not limited to, an alkyl (eg methyl,ethyl), substituted alkyl , alkenyl, substituted alkenyl, acyl, dienyl,arylalkyl, arylalkenyl, aryl, substituted aryl, heterocyclic,substituted heterocyclic, cycloalkyl, substituted cycloalkyl, halo (e.g.Cl, Br, I, F), haloalkyl, nitro, hydroxy, thiol, sulfonyl, carboxy,alkoxy, aryloxy and alkylaryloxy group and the like as would be apparentto one skilled in the art. By alkyl, substitued alkyl, alkenyl andsubstituted alkenyl and the like is meant to encompass straight andbranched molecules, lower (C₁ -C₆) and higher (more than C₆)derivatives. The term "substituted" includes all the substituents setforth above.

In its most preferred embodiment, the protease sensitive peptide is:

    R.sub.1 --X.sub.1 --X.sub.2 --Asn--Asp--R.sub.2

wherein R₁ and R₂ are the same or different and are peptides orpolypeptides and wherein X₁ and X₂ are both Lys residues.

Such a protease sensitive peptide can be placed between the same ordifferent monomers so that upon expression in a suitable host or invitro, the larger molecule can be processed to the peptides locatedbetween the protease sensitive peptides.

The present invention also extends to a nucleic acid molecule comprisinga sequence of nucleotides which encodes or is complementary to asequence which encodes a protease sensitive peptide comprising thesequence:

    --X.sub.1 --X.sub.2 --Asn--Asp--

wherein X₁ and X₂ are preferably the same and are most preferably bothLys residues. Such a nucleic acid molecule may be part of a largernucleotide sequence encoding, for example, a precursor polypeptidecapable of being processed via the protease sensitive sequence intoindividual peptides or monomers.

The protease sensitive peptide of the present invention is particularlyuseful in generating poly and/or multi-valent "precursors" wherein eachmonomer is the same or different and directed to the same or differentactivities such as anti-viral, anti-bacterial, anti-fungal,anti-pathogen and/or anti-predator activity.

Although not wishing to limit this aspect of the invention to any onehypothesis or proposed mechanism of action, it is believed that theprotease acts adjacent the Asn residue as more particularly between theAsn-Asp residues.

The present invention extends to an isolated type II serine PI precursorfrom a plant wherein said precursor comprises at least three PI monomersand wherein at least one of said monomers has a chymotrypsin specificsite and at least one other of said monomers has a trypsin specificsite. Preferably, the PI precursor has four, five or six monomers and isencoded by the nucleic acid molecule as hereinbefore described. Thepresent invention also extends to the individual monomers comprising theprecursor. The present invention also extends to a hybrid recombinant PIprecursor molecule comprising at least two monomers from different Plsas hereinbefore described.

The isolated PI or PI precursor may be in recombinant form and/orbiologically pure. By "biologically pure" is meant a preparation of PI,PI precursor and/or any mixtures thereof having undergone at least onepurification step including ammonium sulphate precipitation, Sephadexchromatography and/or affinity chromatography. Preferably, thepreparation comprises at least 20% of the PI, PI precursor or mixturethereof as determined by weight, activity antibody, reactivity and/oramino acid content. Even more preferably, the preparation comprises30-40%, 50-60% or at least 80-90% of PI, PI precursor or mixturethereof.

The PI or its precursor may be naturally occurring or be a variant asencoded by the nucleic acid variants referred to above. It may alsocontain single or multiple substitutions, deletions and/or additions toits amino acid sequence or to non-proteinaceous components such ascarbohydrate and/or lipid moieties.

The recombinant and isolated PI, PI precursor and mixtures thereof areuseful as laboratory reagents, in the generation of antibodies, intopically applied insecticides as well as orally ingested insecticides.

The recombinant PI or PI precursor may be provided as an insecticidealone or in combination with one or more carriers or other insecticidessuch as the BT crystal protein.

The PI of the present invention is considered to have a defensive rolein organs of the plant, for example, the stigma, against the growth orinfection by pests and pathogens such as fungi, bacteria and insects.There is a need, therefore, to develop genetic constructs which can beused to generate transgenic plants capable of expressing the PIprecursor where this can be processed into monomers of a monomeric PIitself.

Accordingly, another aspect of the present invention contemplates agenetic construct comprising a nucleic acid molecule comprising asequence of nucleotides which encodes or is complementary to a sequencewhich encodes a type II serine PI precursor or monomer thereof from aplant wherein said precursor comprises at least three PI monomers andwherein at least one of said monomers has a chymotrypsin specific siteand at least one of said other monomers has a trypsin specific site andsaid genetic sequence further comprises expression means to permitexpression of said nucleic acid molecule, replication means to permitreplication in a plant cell or, alternatively, integration means, topermit stable integration of said nucleic acid molecule into a plantcell genome. Preferably, the expression is regulated such asdevelopmentally or in response to infection such as being regulated byan existing PI regulatory sequence. Preferably, the expression of thenucleic acid molecule is enhanced to thereby provide greater endogenouslevels of PI relative to the levels in the naturally occurring plant.Alternatively, the PI precursor cDNA of the present invention is usableto obtain a promoter sequence which can then be used in the geneticconstruct or to cause its manipulation to thereby permit over-expressionof the equivalent endogenous promoter. In another embodiment the PIprecursor is a hybrid molecule as hereinbefore described.

Yet another aspect of the present invention is directed to a transgenicplant carrying the genetic sequence and/or nucleic acid molecule ashereinbefore described and capable of producing elevated, enhanced ormore rapidly produced levels of PI and/or PI precursor or hybrid PIprecursor when required. Preferably, the plant is a crop plant or atobacco plant but other plants are usable where the PI or PI precursornucleic acid molecule is expressable in said plant. Where the transgenicplant produces PI precursor, the plant may or may not further processthe precursor into monomers. Alternatively, the genetic sequence may bepart of a viral or bacterial vector for transmission to an insect tothereby control pathogens in insects which would consequently interruptthe transmission of the pathogens to plants.

In still yet another aspect of the present invention, there is providedantibodies to the PI precursor or one or more of its monomers.Antibodies may be monoclonal or polycional and are useful in screeningfor PI or PI precursor clones in an expression library or for purifyingPI or PI precursor in a fermentation fluid, supernatant fluid or plantextract.

The genetic constructs of the present invention can also be used topopulate the gut of insects to act against the insect itself or anyplant pathogens therein or to incorporate into the gut of animals tofacilitate the digestion of plant material.

The present invention is further described by reference to the followingnon-limiting Figures and Examples.

In the Figures:

FIG. 1 shows the nucleic acid sequence (SEQ ID NO. 2) of the pNA-PI-2insert and the corresponding amino acid sequence (SEQ ID NO. 3) of theN. alata PI protein. The amino acid sequence is numbered beginning with1 for the first amino acid of the mature protein. The signal sequence isencoded by nucleotides 1 to 97 and the amino acid residues have beenassigned negative numbers. The reactive site residues of the inhibitorare boxed. The N. alata PI sequence contains six similar domains (domain1, residues 1 to 58, domain 2, residues 59-116, domain 3, residues117-174, domain 4, residues 175-232, domain 5, residues 233-290 anddomain 6, residues 291-343).

FIG. 2 is a photographic representation showing a gel blot analysis ofRNA from various organs of N. alata Gel Blot of RNA isolated from organsof N. alata and from stigmas and styles of N. tabacum and N. sylvestris,hybridised with the cDNA clone NA-PI-2. St, stigma and style; Ov,ovaries; Po, pollen; Pe, petals; Se, sepals; L, non-wounded leaves; L4,leaves 4 h after wounding; L24, leaves 24 h after wounding; Nt, N.tabacum stigma and style; Ns, N. sylvestris stigma and style; Na HindIIIrestriction fragments of Lambda-DNA.

The NA-PI-2 clone hybridised to 2 mRNA species (1.0 and 1.4 kb). Thelarger mRNA was predominant in stigma and styles, whereas the smallermRNA species was more dominant in other tissues. After high stringencywashes, the 1.0 kb mRNA from stigma and style no longer hybridises tothe NA-PI-2 probe.

FIG. 3 is a photographic presentation depicting in situ localisation ofRNA homologous to NA-PI-2 in stigma and style.

(a) Autoradiograph of a longitudinal cryosection through the stigma andstyle of a 1 cm long bud after hybridisation with the ³² P-labelledNA-PI-2 cDNA probe.

(b) The same section as (a), stained with toluidine blue. c, cortex; v,vascular bundles; tt, transmitting tract; s, stigmatic tissue.

The cDNA probe labelled the cells of the stigma heavily and somehybridisation to the vascular bundles can be seen. There was nohybridisation to the epidermis, cortical tissue or transmitting tissue.Scale bars=200 μm.

FIG. 4 is a photographic representation of a gel blot analysis ofgenomic DNA of N. alata Gel blot analysis of N. alata genomic DNAdigested with the restriction enzymes EcoRI or HindIII, and probed withradiolabelled NA-PI-2. Size markers (kb) are HindIII restrictionfragments of Lambda-DNA.

EcoRI produced two hybridising fragments (11 kb and 7.8 kb), whileHindIII gave three large hybridising fragments (16.6, 13.5 and 10.5 kb).The NA-PI-2 clone appears to belong to a small multigene familyconsisting of at least two members.

FIG. 5 is a graphic representation of PI activity in various organs ofN. alata Buffer soluble extracts from various organs were tested fortheir ability to inhibit trypsin and chymotrypsin. Stigma and sepalextracts were the most effective inhibitors of both trypsin (A) andchymotrypsin (B).

FIG. 6 depicts the steps of the purification of PI from N. alatastigmas.

(a) Sephadex G-50 gel filtration chromatography of ammonium sulphateprecipitated proteins from stigma extracts. The PI activity eluted latein the profile.

(b) 20% w/v SDS-polyacrylamide gel (Laemili, 1970) of fractions acrossthe gel filtration column. The gel was silver stained and molecularweight markers (Pharmacia peptide markers) are in kilodaltons. A proteinof about 6 kD (arrowed) coelutes with the proteinase inhibitor activity.

(c) Analysis of PI-containing fractions at different stages of thepurification procedure, by SDS-PAGE. Lane 1, crude stigma extract (5μg); Lane 2, stigma proteins precipitated by 80% w/v ammonium sulphate(5 μg); Lane 3, PI protein eluted from the chymotrypsin affinity column(1 μg).

The PI is a 6 kD protein and is a major component in unfractionatedbuffer soluble extracts from stigmas.

FIG. 7 is a graphical representation showing hydropathy plots of the PIproteins encoded by the NA-PI-2 clone from N. alata and the potato andtomato PI II cDNAs. Values above the line denote hydrophobic regions andvalues below the line denote hydrophilic regions. The putative signalpeptides are shaded. The hydrophobicity profile was generated using thepredictive rules of Kyte and Doolittle (1982) and a span of 9consecutive amino acids.

(a) Hydropathy profile of the N. alata PI protein The six repeateddomains in the predicted precursor protein are labelled I-VI. Thehydrophilic regions containing the putative cleavage sites forproduction of the 6 kD PI species are arrowed. The regions correspondingto the peptides that would be produced by cleavage at these sites aremarked C for chymotrypsin inhibitor, T for trypsin inhibitor and x forthe two flanking peptides.

(b) Hydropathy profile of the potato PI II protein. (Sanchez-Serrano etal., 1986). The two repeated domains in the PI II protein are labelled Iand II. The putative cleavage sites for production of PCI-1 are arrowed(Hass et al., 1982) and the region spanned by PCI-1 is marked.

c) Hydropathy profile of the polypeptide encoded by the tomato PI IIcDNA (Graham et al., 1985). The two domains are labelled, I and II andthe residues which would be potential processing sites are arrowed.These sites are not present in regions predicted to be hydrophilic andconsequently a cleavage product is not marked.

FIG. 8 shows an immunoblot analysis of the PI protein in stigmas ofdeveloping flowers.

(a) Developing flowers of N. alata.

(b) SDS-PAGE of stigma proteins at the stages of development shown in(a) 5 μg of each extract was loaded. The peptide gel was silver stainedand molecular weight markers (LKB Low Molecular weight and Pharmaciapeptide markers) are in kilodaltons.

(c) Immunoblot of a gel identical to (b), probed with anti-PI antiserum.

Stigmas from developing flowers contain four proteins of approximately42 kD, 32 kD, 18 kD and 6 kD that bind to the anti-PI antibody. The 42kD and the 18 kD components decrease in concentration as the flowersmature, while the 6 kD PI protein reaches a maximum concentration justbefore anthesis. The level of the 32 kD component, which runs as adoublet, does not alter significantly during flower development.

FIG. 9 shows the separation and identification of the 6 kD proteinaseinhibitor species from N.alata stigmas

A. Separation of the 6 kD PIs by reversed phase HPLC chromatography Fourmajor peaks were obtained with retention times of about 15.5 min(peak1),20.5 min(peak2), 22.5 min(peak3), 24 min(peak4). The peptides in eachpeak have been identified by a combination of N-terminal analysis andmass spectrometry. See B for description of C1 and T1-T4.

B. The five homologous peptides produced from the PI precursor protein:C1, chymotrypsin inhibitor, T1-T4 trypsin inhibitors. The solid barsrepresent the reactive sites of the inhibitors. The precursor protein isdrawn minus the signal sequence. region of the six repeated domains(amino acids 1-343, FIG. 1). non-repeated sequence (amino acids 344-368,FIG. 1). The arrows point to the processing sites in the precursorprotein.

C. The amino acid sequence of C1 and T1-T4 predicted from the cDNA cloneand confirmed by N-terminal sequencing of the purified peptides. Theamino acid at the carboxy-terminus of each peptide was obtained byaccurate mass determination using an electro-spray mass spectrometer.The C1 and T1 inhibitors differ by five amino acids (bold). Two of theseamino acids are located at the reactive site (underlined) and the othertwo to three reside at the carboxy-terminus. Peptides T2-T4 have changesin three amino acids (boxed) that are conserved between C1 and T1.Peptides T2 and T3 are identical to each other. Mass spectrometry wasused to demonstrate that other forms of C1 and T1-T4 occur due tonon-precise trimming at the N- and C-termini. That is, some forms aremissing residue 1 or residue 53 and others are missing both residue 1and 53 (see Table 2).

FIG. 10 shows the amino-acid sequence around the processing sites in theprecursor PI protein.

The sequence in bold is the amino-terminal sequence obtained from thepurified PI protein. The sequence labelled with negative numbers is theflanking sequence predicted from the cDNA clone. The predicted precursorprotein contains-six repeats of this sequence.

FIG. 11 shows the PI precursor produced in a baculovirus expressionsystem and the products obtained after digestion of the affinitypurified PI precursor by the endoproteinase Asp-N.

A. The PI precursor produced by the recombinant baculovirus.

Imunoblot containing affinity and HPLC purified PI precursor fromN.alata stigmas at the green bud stage of development (lane 1) andaffinity purified PI precursor produced by the recombinant baculovirus(lane 2). Proteins were fractionated by electrophoresis on a 15% w/vSDS-polyacrylamide gel prior to electrophoretic transfer tonitrocellulose. The blot was incubated with the antibody raised inrabbits to the 6 kD PI species from stigmas. The recombinant virusproduced an immunorective protein of 42 kD that is the same size as thePI precursor protein produced by stigmas (arrowed).

B. Cleavage of the PI precursor by endoproteinase Asp-N.

15% SDS-polyacrylamide gel stained with silver containing: 1, PIprecursor, produced by baculovirus, incubated without enzyme. 2, enzymeincubated without precursor. 6 kD, PI peptides of about 6 kD purifiedfrom N. alata stigmas. 1 m, 5 m, 30 m, reaction products produced after1, 5 and 30 minutes of incubation. 2 h and 24 h, reaction products after2 and 24 h of incubation. Peptides of about 6-7 kD were detected withinone minute of incubation of the precursor with the enzyme. After 24 honly peptides of 6-7 kD were detected. The bands smaller than 42 kD intrack 1 are due to truncated forms of the precursor produced bypremature termination of translation in the baculovirus expressionsystem.

FIG. 12 Preparative chromatography by reversed phase HPLC of thepeptides produced from the precursor by Asp-N digestion

HPLC profile of peptides produced by Asp-N digestion of the PIprecursor. The major peaks had a retention time of 19 min (termedAsp-N1) and 21 min (termed Asp-N2). The peptides in these peak fractions(1 & 2) had a slightly slower mobility on SDS-PAGE than the 6 kDpeptides from stigmas (C, inset). The proteinase inhibitory activity ofAsp-N1 and Asp-N2 was tested against trypsin and chymotrypsin.

FIG. 13 shows a comparison of the trypsin and chyrnotrypsin inhibitionactivity of the PI precursor, PI peptides from stigmas and in vitroproduced PI peptides from the PI precursor.

PI precursor or PI peptides (0-1.0 μg) were tested for their ability toinhibit 1.0 μg of trypsin or chymotrypsin as described in the materialsand methods. Inhibitory activity is expressed as the percentage ofproteinase activity remaining after the proteinase had been preincubatedwith the PI with 100% remaining activity taken as the activity of theproteinase preincubated with no PI. Experiments were performed induplicate and mean values were plotted. Deviation from the mean was 8%or less.

FIG. 14 is a graphical representation showing a growth curve for T.comnodus nymphs reared on control artificial diet, soybean Bowman-Birkinhibitor and N. alata PI. The vertical axis represents the mean weightof the crickets in each treatment (+/-standard error) in mg. Thehorizontal axis represents the week number. The crickets reared on theN. alata PI showed a lower mean weight than those reared on both thecontrol diet and the diet containing the soybean inhibitor, throughoutthe experiment.

    ______________________________________                                        SUMMARY OF SEQ ID. NOs                                                        ______________________________________                                        SEQ ID NO. 1                                                                             Nucleotide coding region of N. alata PI precursor                    SEQ ID NO. 2 Full length nucleotide sequence of N. alata PI                    precursor                                                                    SEQ ID NO. 3 Amino acid sequence corresponding to SEQ ID                       NO. 1                                                                        SEQ ID NO. 4 Residues 1-24 of SEQ ID NO. 2 (peptide 1)                        SEQ ID NO. 5 Residues 25-82 of SEQ ID NO. 2 (peptide 2)                       SEQ ID NO. 6 Residues 83-140 of SEQ ID NO. 2 (peptide 3)                      SEQ ID NO. 7 Residues 141-198 of SEQ ID NO. 2 (peptide 4)                     SEQ ID NO. 8 Residues 199-256 of SEQ ID NO. 2 (peptide 5)                     SEQ ID NO. 9 Residues 257-314 of SEQ ID NO. 2 (peptide 6)                     SEQ ID NO. 10 Residues 315-368 of SEQ ID NO. 2 (peptide 7)                    SEQ ID NO. 11 N-terminal amino acid sequence of 6 kD PI protein                         SEQ ID NO. 12 N-terminal amino acid sequence of 6 kD PI                      protein                                                            ______________________________________                                    

EXAMPLE 1 1. MATERIALS AND METHODS

Plant Material

Nicotiana alata (Link et Otto) plants of self-incompatibility genotypeS₁ S₃, S₃ S₃ and S₆ S₆ were maintained under standard glasshouseconditions as previously described (Anderson et al., 1989). Organs werecollected directly into liquid Nitrogen to avoid induction of a woundresponse and stored at -70° until required. To study the effect ofwounding on gene expression, leaves were wounded by crushing across themid-vein with a dialysis clip. Leaves were collected 4 and 24 hoursafter wounding.

Identification and Sequencing of a cDNA Clone Encoding PI

Polyadenylated RNA was prepared fron stigmas and styles, isolated frommature flowers of N. alata (genotype S₃ S₃), and used to construct acDNA library in Lambda gt10 (Anderson et al., 1989). Single stranded ³²P-labelled cDNA was prepared from mRNA from stigmas and styles of N.alata (genotype S₃ S₃ and S₆ S₆) and used to screen the library forhighly expressed clones which were not S-genotype specific (Anderson etal., 1989). Plaques which hybridised strongly to cDNA probes from bothS-genotypes were selected and assembled into groups on the basis ofcross-hybridisation. The longest clone of each group was subcloned intoM13mp18 and pGEM 3zf +, and sequenced using an Applied Biosystems Model373A automated sequencer. Both dye primer and dye terminator cyclesequencing chemistries were performed according to standard AppliedBiosystems protocols. Consensus sequences were generated using SeqEd™sequence editing software (Applied Biosystems). The GenBank database wassearched for sequences homologous to these clones. Because of the highdegree of sequence similarity between the six domains of the N. alata PIclone, sequencing primers were made to non-repeated 3' sequences(nucleotides 1117-1137, 1188-1203 and 1247-1267), and to a 5' sequencebefore the start of the repetitive regions (nucleotides 74-98). Inaddition, the pNA-PI-2 insert was restricted with endonuclease HaeIII,which cut at nucleotides 622 and 970 to produce three fragments. Thefragments were subcloned into pGEM7zf+ and sequenced in both directions,using the M13 forward and reverse primers. The repetitive nature of thepNA-PI-2 insert rendered it unstable in both phagemid and plasmidvectors when cultures were grown longer than 6 hours.

RNA Gel Blot Analysis

Total RNA was isolated and separated on a 1.2% w/v agarose/formaldehydegel as previous described (Anderson et al., 1989). The RNA wastransferred to Hybond-N (Amersham) and probed with the insert frompNA-PI-2 labelled with ³² P using random hexanucleotides (1×10⁸ cpm μg⁻¹; 1×10⁷ cpm ml⁻¹)(Feinberg and Vogelstein, 1983). Prehybridisation andhybridisation, at 68° C., were as described by Anderson et al. (1989).The filters were washed in 2×SSC, 0.1% w/v SDS or 0.2×SSC, 1% w/v SDS at68° C.

In Situ Hybridisation

In situ hybridisation was performed as described by Cornish et al.,1987. The probe was prepared by labelling the insert from pNA-PI-2 (100ng) to a specific activity of 10⁸ cpm μg⁻¹ by random hexanucleotidepriming (Feinberg and Vogelstein, 1983). The labelled probe wasprecipitated, and resuspended in hybridisation buffer (50 μl), and 5 μlwas applied to the sections. The sections were covered with coverslips,and incubated overnight at 40° C. in a closed box containing 50% v/vformamide. After incubation, sections were washed sequentially in 4×SSCat room temperature, 2×SSC at room temperature, and 1×SSC at 40° C. for40 min. The slides were dried and exposed directly to X-ray film (CronexMRF 32, Dupont) at room temperature, overnight. Hybridised sections werecounterstained with 0.025% w/v toluidine blue in H₂ O, and mounted inEukitt (Carl Zeiss, Freilburg, FRG). Autoradiographs were transposedover sections to give the composites shown.

DNA Gel Blot Analysis

Genomic DNA was isolated from young leaves of N. alata by the procedureof Bernatzky and Tanksley (1986). DNA (10 μg) was digested to completionwith the restriction endonucleases EcoRI or HindIII, separated byelectrophoresis on a 0.9% w/v agarose gel, and transferred to Hybond-N(Amersham) by wet blotting in 20×SSC. Filters were probed and washed asdescribed for RNA blot analysis.

Preparation of Protein Extracts

Soluble proteins were extracted from plant material by freezing thetissue in liquid N₂, and grinding to a fine powder in a mortar andpestle. The powdered tissue was extracted in a buffer consisting of 100mM Tris-HCl, pH 8.5, 10 mM EDTA, 2 mM CaCl₂, 14 μM β-mercaptoethanol.Insoluble material was removed by centrifugation at 10,000 g for 15 min.Protein concentrations were estimated by the method of Bradford (1976)with Bovine Serum Albumin (BSA) as a standard.

Proteinase Inhibition Assays

Protein extracts and purified protein were assayed for inhibitoryactivity against trypsin and chyrnotrypsin as described by Rickauer etal. (1989). Inhibitory activity was measured against 1 pg of trypsin(TPCK-treated; Sigma) or 3 μg of chymotrypsin (TLCK-treated; Sigma). Therate of hydrolysis of synthetic substrates N-α-P-tosyl-L-arginine methylester (TAME) and N-benzoyl-L-tyrosine ethyl ester (BTEE) by trypsin andchymotrypsin, respectively, were taken as the uninhibited activity ofthe enzymes. Inhibitory activity of the extract was expressed as thepercentage of control protease activity remaining after the protease hadbeen pre-incubated with the extract. The PI peptides from stigma, PIprecursor and Asp-N processed peptides were assayed for inhibitoryactivity as described by Christeller et al. (1989).

Purification of the N. alata PI Protein

Stigmas (1000; 10 g) were ground to a fine powder in liquid N₂, andextracted in buffer (100 mM Tris-HCl, pH8.5, 10 mM EDTA, 2 mM CaCl₂, 14μM β-mercaptoethanol, 4 ml/g tissue). To concentrate the extract priorto the first purification step, gel filtration, the inhibitory activitywas precipitated with 80% w/v ammonium sulphate, the concentrationrequired to precipitate all the proteinase inhibitory activity.

The ammonium sulphate pellet was resuspended in 5 ml of 0.15 M KCl , 10mM Tris-HCl, pH 8.1, and loaded onto a Sephadex G-50 column (2 cm×100cm) equilibrated with the same buffer. The fractions (10 ml) eluted fromthis column and containing proteinase inhibitory activity were pooledand applied to an affinity column of Chymotrypsin-Sepharose CL4B [100 mgTLCK-treated α-chymotrypsin (Sigma) cross-linked to 15 ml Sepharose CL4B(Pharmacia) by manufacturers instructions]. The column was washed with10 volumes of 0.15M KCl/10 mM Tris-HCl, pH 8.1, prior to elution ofbound proteins with 7 m urea, pH 3 (5 ml fractions). The eluate wasneutralised immediately with 200 μl 1M Tris-HCl pH 8, and dialyzedextensively against deionised H₂ O.

Amino Acid Sequence Analysis

Purified PI protein was chromatographed on a reverse phase HPLCmicrobore column prior to automated Edman degradation on a gas phasesequencer (Mau et al., 1986). Phenylthiohydantoin (PTH) amino acids wereanalysed by HPLC as described by Grego et al. (1985).

Production of a Polyclonal Antiserum to the N. alata PI

The purified proteinase inhibitor (FIG. 6c, lane 3) was conjugated to acarrier protein, keyhole limpet haemocyanin (KLH) (Sigma), usingglutaraldehyde, as follows. 1 mg of PI protein was dissolved in 1.5 mlH₂ O, and mixed with 0.3 mg KLH in 0.5 ml of 0.4M phosphate buffer,pH7.5. 1 ml of 20 mM glutaraldehyde was added dropwise over 5 min, withstirring at room temperature. The mixture was stirred for 30 min at roomtemperature, 0.25 ml of glycine was added, and the mixture was stirredfor a further 30 min. The conjugated protein was then dialyzedextensively against normal saline (0.8% w/v NaCl). The equivalent of 100μg of PI protein was used for each injection. Freund's complete adjuvantwas used for the first injection, and incomplete adjuvant for twosubsequent booster injections. The IgG fraction of the antiserum wasseparated on Protein A Sepharose (Pharmacia) according to manufacturer'sinstructions.

Protein Gel Blot Analysis

Protein extracts were electrophoresed in 15% w/v SDS-polyacrylamide gels(Laemmli, 1970) and transferred to nitrocellulose in 25 mM Tris-HCl, 192mM glycine, 20% v/v methanol, using a BioRad Trans-Blot®Semi-dryelectrophoretic transfer cell (12 V, 20 min). Loading and proteintransfer were checked by staining the proteins on the membranes withPonceau S (Harlow and Lane, 1988). Membranes were blocked in 3% w/vbovine serum albumin for 1 h, and incubated with the anti-PI antibody (2μg/ml in 1% w/v BSA, Tris Buffered Saline) overnight at roomtemperature. Bound antibodywas detected using biotinylated donkeyanti-rabbit IgG (1/500 dilution, Amersham) and the AmershamBiotin-Streptavidin system according to procedures recommended by themanufacturer.

Proteolysis of the PI Precursor by Endoproteinase Asp-N

Affinity-purified PI precursor (1.25 mg) was incubated at 37° C. withendoproteinase Asp-N (2 μg) in 100 mM NH₄ HCO₃, pH 8.5 in a total volumeof 1 ml for 48 h. Reaction products were separated by reversed-phaseHPLC using an analytical Brownlee RP-300 Aquapore column (C8, 7 μm,4.6×100 mm). The column was equilibrated in 0.1% v/v TFA and peptideswere eluted with the following program: 0-25%B (60% v/v acetonitrile in0.089% v/v TFA) applied over 5 min, followed by a gradient of 25-42%Bover the next 40 min, and ending with a gradient of 42-100%B over 5minutes. The flow rate was 1.0 ml/min and peptides were detected byabsorbance at 215 nm. Each peak was collected manually and freeze dried.Concentration was estimated by response obtained with each peak on theUV detector at 215 nm.

2. CLONING OF PI PRECURSOR GENE

Isolation and Characterisation of the PI cDNA Clone

A cDNA library, prepared from mRNA isolated from the stigmas and stylesof mature flowers of N. alata was screened for clones of highlyexpressed genes which were not associated with self-incompatibilitygenotype. Clones encoding a protein with some sequence identity to thetype II proteinase inhibitors from potato and tomato (Thornburg et al.,1987; Graham et al., 1985) were selected. The largest clone, NA-PI-2, is1360 base pairs long with an open reading frame of 1191 nucleotides. Thenucleic acid sequence (SEQ ID NO. 2) and the predicted amino acidsequence (SEQ ID NO. 3) of the N. alata clone, NA-PI-2 is shown inFIG. 1. There are no potential N-glycosylation sites.

Surprisingly, the N. alata cDNA clone encodes a protein with sixrepeated domains that have high, but not perfect, sequence identity(FIG. 1). Each of these domains contains a potential reactive site whichis highlighted in FIG. 1. The residues at the putative reactives sitesof the N. alata PI are consistent with the inhibitor having two siteswhich would specifically inhibit chymotrypsin (Leu5-Asn6, Leu63-Asn64)and four sites specific for trypsin (Arg121-Asn122, Arg179-Asn180,Arg237-Asn238 and Arg295-Asn296).

To ensure that the repeat structure of NA-PI-2 was not due to a cloningartifact, three additional cDNA clones were sequenced, and found to beidentical to NA-PI-2.

Table 1 is a comparison of the percentage amino acid identity of the sixdomains of the PI precursor.

Temporal and Spatial Expression of the PI mRNA

Total RNA, isolated from various tissues of N. alata, was probed withthe PI cDNA clone in the RNA gel blot analyses shown in FIG. 2. Twohybridising messages of 1.0 and 1.4 kb were present in total RNAisolated from styles (including stigmas). Only the larger message, whichwas predominant in this tissue, is of sufficient size to encode the cDNAclone NA-PI-2 (1.4 kb). The smaller message is not detected with thecDNA probe at higher stringency. An homologous message of approximately1.4 kb was also present in RNA isolated from the styles of N. tabacumand N. sylvestris (FIG. 2).

In the other floral organs (except pollen), both messages weredetectable at low levels, however, the smaller RNA species appeared moreabundant. There was no hybridisation to pollen RNA. No hybridisingspecies were evident in leaf RNA, but two species, 1.0 and 1.4 kb weredetected 24 hours after mechanical wounding. The smaller message (1.0kb) was more abundant in this case.

In situ hybridisation of radiolabelled N. alata PI cDNA to longitudinalsections of styles from immature (1 cm long) buds is shown in FIG. 3.RNA homologous to the cDNA clone bound strongly to cells of the stigmaand weakly to vascular bundles. No hybridisation was detected in thecortical tissue, transmitting tract tissue, or epidermis of the style.The same pattern of hybridisation was observed in mature receptiveflowers. Control sections treated with ribonuclease A prior tohybridisation were not labelled.

Genomic DNA Blot Analysis

The cDNA clone NA-PI-2, was used as a probe on the DNA gel blot shown inFIG. 4 which contained genomic DNA, digested with either EcoRI orHindIII. EcoRI produced two hybridising fragments (11 kb and 7.8 kb) andHindIII produced three large hybridising fragments (16.6, 13.5 and 10.5kb).

Distribution of PI Activity in Various Tissues of N. alata

The inhibition of trypsin and chymotrypsin by crude extracts of variousorgans of N. alata is shown in FIG. 5. Stigma extract was the mosteffective inhibitor of both trypsin and chymotrypsin. The stigmaextracts had up to eight times more inhibitory activity than sepalextracts, and more than 20 times more activity than extracts fromstyles, petals, leaves and wounded leaves.

Purification of PI from N. alata Stigmas

Stigmas of N. alata were extracted in buffer and the inhibitory activitywas concentrated by precipitation with 80% w/v ammonium sulphate. Theprecipitate was redissolved and fractionated by gel filtration onSephadex G-50. Most of the protein in the extract eluted early in theprofile illustrated in FIGS. 6a and 6b, relative to the proteinaseinhibitor. Fractions with proteinase inhibitor activity were pooled andapplied to an affinity column of chymotrypsin-Sepharose. The PI activityco-eluted with a protein of about 6 kD, which appeared to migrate as asingle band on the 20% SDS-polyacrylamide gel shown in FIG. 6c. Thepurity of the PI at various stages of purification was assessed bySDS-PAGE (FIG. 6c). The purified inhibitor represented approximately 50%of the inhibitory activity present in the crude extract.

Amino Acid Sequence of the N-terminus of the 6 kD PI Protein

The N-terminal amino acid sequence DRICTNCCAG(T/K)KG (SEQ ID NO. 11; SEQID NO. 12, respectively) was obtained from the purified PI protein. Thissequence of amino acids corresponds to six regions in the deducedsequence of the cDNA clone, starting at positions 25, 83, 141, 199, 257and 315 in FIG. 1. At position 11 of the N-terminal sequence, boththreonine and lysine were detected.

This is consistent with the purified inhibitor comprising a mixture ofsix peptides beginning with the sequences underlined in FIG. 1, as thefirst two peptides contain threonine at this position, while the otherfour peptides have lysine at this position. The position of thesepeptides relative to the six repeated domains in the predicted precursorprotein is illustrated in FIG. 7. Five of the six predicted 6 kDpeptides, contain a reactive site for either chymotrypsin or trypsin(FIG. 1 and 7). The sixth potential peptide is four amino-acids shorterthan the other five peptides (fifty eight amino-acids) and may not beactive, as it does not contain an inhibitory site. The peptide from theN-terminus (x in FIG. 7) has a potential chymotpsin reactive site but ismuch shorter (24 amino acids).

Distribution of the PI Protein in N. alata

A polyclonal antiserum was raised to the purified PI protein conjugatedto keyhole limpet haemocyanin. The antibody reacted strongly with thepurified 6 kD PI protein in immunoblot analyses and bound only to a 6 kDand a 32 kD protein, which appears as a doublet, in total stigma andstyle extracts from mature flowers. FIG. 8 is an immunoblot containingprotein extracts of stigmas from flowers at different stages ofdevelopment (1 cm long buds to mature flowers) probed with the anti-PIantiserum. Larger cross reacting proteins of approximately 18 kD, and 42kD were detected in buds from 1 cm to 5 cm in length in addition to the6 kD and the 32 kD protein. The 18 kD and 42 kD proteins decreased inconcentration with maturity, while the 6 kD protein reached a peakconcentration just before anthesis. The concentration of the 32 kDprotein remained relatively constant during flower maturation.

                  TABLE 1                                                         ______________________________________                                        N. alata PI                                                                                1     2        3   4      5   6                                  ______________________________________                                        N. alata                                                                              1              100    88  88     90  79                                  2   88 88 90 79                                                               3    97 95 86                                                                 4     98 90                                                                   5      90                                                                     6                                                                          ______________________________________                                    

EXAMPLE 2 PURIFICATION AND IDENTIFICATION OF PI MONOMERS 1. MATERIALSAND METHODS

Separation of the 6 kD PI Species by Reversed Phase Chromatography

Stigmas (21,000) were ground and extracted as described for purificationof the PI protein. After gel filtration on a Sephadex G-50 gelfiltration column (5 cm×800 cm, 3000 stigmas per separation) thepeptides were lyophilized and applied to a Brownlee RP-300 C8Reversed-phase column, 10×250 mm, on a Beckman HPLC system Gold, andeluted with 0.1% v/v Trifluoroacetic acid (TFA) and an acetonitrilegradient (0-10% over 5 mins, 10-25% over 40 mins and 25-60% over 10mins), at 5 ml/min. Peak fractions, designated fraction 1, 2, 3 and 4were collected and freeze dried.

Electrospray Mass Spectrometry

On line mass spectrometric analysis of HPLC eluates was performed byapplication of 20 pmoles of each PI preparation (fraction 1, 2, 3 & 4)in 2 μl of water onto a Brownlee RP-300 C8 reversed-phase column(150×0.20 mm internal diameter fused-silica capillary column) on amodified Hewlett-Packard model HP1090L liquid chromatograph and elutionwith a linear gradient of acetonitrile (0.05% v/v TFA to 0.045% v/vTFA/60% v/v acetonitrile in 30 min.) at a flow rate of 1 μl/min and acolumn temperature of 25° C. The eluant was monitored at 215 nm using aSpectral Physics forward optics scanning detector with a 6-mm pathlengthU-shaped axial beam capillary flow cell (LC Packings, Netherlands). Massspectra were acquired on a Finnigan-Mat triple quadrupole massspectrometer (modelTSQ-700, San Jose, Calif.) equipped with anelectrospray ionisation (ESI) source (Analytica, Branford, Conn.). Theelectrospray needle was operated in positive ion mode at a voltagedifferential of -4 kV. The sheath liquid was 2-methoxyethanol deliveredat 1 μl/min via a syringe drive (Harvard Apparatus, South Natick,Mass.). The nitrogen drying gas conditions were as follows: heatertemperature, 275° C.; pressure, 15 psi; flow rate, ˜15 stdL/min. Thenitrogen sheath gas was supplied at 33 psi. Gaseous nitrogen wasobtained from a boiling liquid nitrogen source. Peptides were introducedinto the ESI source at 1.0 μl/min by on-line capillary RP-HPLC asdescribed above. Spectra were acquired scanning from m/z 400 to 2000 ata rate of 3 sec. Data collection and reduction were performed on aDec5100 computer using Finnigan BIOMASS™ software.

2. RESULTS

Separation and Identification of the Individual 6 kD PI Species fromN.alata Stigmas

The five of the six peptides of about 6 kD that were predicted to bepresent in the purified 6 kD PI preparation have been separated fromeach other by reversed-phase HPLC chromatography. Four peaks wereobtained (FIG. 9a) and the peptides within each peak were identified byelectrospray mass spectrometry (Table 2). The peptides have beendesignated C1, T1, T2, T3 and T4 according to their position in the PIprecursor and the presence of a chymotrypsin or trypsin reactive site(FIG. 9b). The first HPLC peak (FIG. 9a) corresponds to the chymotrypsininhibitor C1, the second peak is composed of a mixture of T2 and T3(identical to each other) and T4 that differs from T2 and T3 by oneamino-acid at position 32. The third peak contains the peptide T1 andthe fourth peak is composed of a nixture of T1, T2/T3 and T4 (Table 2).

The site of processing has not been precisely determined, but is likelyto be located between the aspartate (N) and asparagine (D) residues inthe sequence outlined in FIG. 10. Proteases with specific requirementsfor asparagine residues have been isolated from vacuoles from immaturesoybean seeds and pumpkin cotyledons (Scott et al., 1992, Hara-Nishimuraet al., 1991). This is consistent with the immunogold localization ofthe PI in the vacuoles of the papillae and the underlying secretorycells in the stigma of N.alata (Atkinson, 1992). In the case of theN.alata PI, processing analogous to that of peptide hormones is alsopossible because each of the possible 6 kD peptides are flanked bydibasic residues (Lys-Lys, position-2 & -3 in FIG. 10). However, asystem like this has not been described in plants, and it is more likelythat the dibasic residues contribute to the predicted hydrophilic loopsthat present the processing site on the surface of the molecule.

The data from the mass spectrometric analysis shows that once theinitial cleavage has occurred the new carboxy terminus is trimmed back(FIG. 10). The EEKKN sequence (SEQ ID NO. 14) is removed completely butthe trimming is not precise, sometimes an additional amino acid isremoved. Steric hindrance probably prevents further trimming.Occasionally the aspartate is also removed from the N-terminus.

EXAMPLE 3

Production of PI Precursor in Insect Cell (Sf9) Culture Using aRecombinant Baculovirus Vector.

cDNA encoding the PI precursor (FIG. 1) was inserted into the Eco R1site of the plasmid vector pVL 1392, which is the sane as pVL941(Lucknow and Summers, 1989) except that a multiple cloning site wasinserted at the BamH1 site. The plasmid designated pRH11, contains thePI cDNA in the correct orientation with respect to the direction oftranscription directed by the polyhedrin promoter. Recombinantbaculovirus was obtained by co-transfection of Spodoptera frugiperdacells with baculovirus DNA and pRH11. The recombinant viruses, producedby homologous recombination, were plaque purified and amplified prior toinfection of insect cells for protein production. All procedures forproduction of recombinant baculovirus, titration of the virus andmaintenance and infection of the Sf9 cells were obtained from King andPosse (1992). For production of the PI precursor, monolayers of Sf9cells in large flasks (175 cm²) were infected at the time of confluencewith an inoculum of high-titre recombinant virus at a multiplicity ofinfection of 5-10 pfu/cell. Culture fluid was collected after 4 days ofinfection, clarified by centrifugation and the PI precursor was purifiedby application to a Chymotrypsin-Sepharose affinity column as describedfor the 6 kD PI species from stigmas. PI precursor eluted from thecolumn in 7M urea, pH3 was neutralized immediately with 1M Tris-HClbuffer pH8, dialysed extensively against Milli-Q water, concentrated20-50 fold by ultrafiltration using a Diaflow YM10 filter and storedfrozen at -20° C.

The cDNA clone encoding the PI precursor was engineered into abaculovuirus vector for the production of the precursor from infectedinsect cells. The insect cells produced a 42 kD protein that crossreacted with the antibodies raised to the 6 kD PI peptides from stigmaand bound to the chymotrypsin affinity column. This 42 kD protein wasidentical in size to the 42 kD precursor produced in the immaturestigmas of N.alata (FIG. 11) and had the N-terminal sequenceLysAlaCysThrLeuAsn (SEQ ID NO. 13) demonstrating that the signalsequence had been processed correctly by the insect cells (FIG. 1).Based on these results, the 42 kD protein produced in the baculovirusexpression system will now be referred to as the PI precursor. The 42 kDPI precursor had inhibitory activity against chymotrypsin but noinhibitory activity against trypsin (FIG. 13). Processing of the PIprecursor by the endoproteinase AspN led to the production of stablepeptides of about 6 kD that were partially purified by reversed phaseHPLC (FIG. 12). These peptides have equivalent inhibitory activityagainst trypsin and chymotrypsin as the 6 kD peptides isolated fromstigma, indicating that processing of the precursor is required toactivate the trypsin inhibitory activity but not all the chymotrypsinactivity. Since AspN cleaves specifically adjacent to Aspartate residues(between Asn-1 and Asp1 in FIG. 10) and has no trimming activity, thepeptides produced in vitrowill be similiar to those produced in stigmasexcept for the presence of the sequence EEKKN (SEQ ID NO. 14) at theC-terminus. This provides further evidence that precise processing ofthe N-and C-termini is not required to obtain an active 6 kD PI peptide.Asp-N1 is more efficient at inhibiting chymotrypsin than trypsin and isthus likely to be predominantly a C1 analogue (FIG. 9b). Asp-N2 is amore efficient trypsin inhibitor and probably contains the T1-T4analogues.

EXAMPLE 4

Effect of PIs on Protease Activity in Unfractionated Gut Extacts fromVarious Insects

Activity of PIs on gut proteases was measured using the procedure ofChristeller et al., (1992) as follows. An aliquot of 1 uM of inhibitor(0-10 μl, at least 5-fold excess over proteases present in the gut) wasmixed with 150 μl of 10 mM CAPS buffer, pH 10, and preincubated witheach insect gut extract (0-15 μl ), for 20 min at 30° C. The reactionwas started by the addition of 50 μl of ¹⁴ C -labelled casein substrate(400 μg protein, specific activity 25,000-75,000 dpm mg⁻¹) and continuedfor 30 min at 30° C. until 50 μl of cold 30% (w/v) TCA was added toterminate the reaction. After incubation on ice for 30 min, undigestedprotein was pelleted by centrifugation at 20° C. for 5 min at 10,000 g.The supernatant was removed, mixed with scintillation fluid and theradioactivity measured. Assays were performed at pH 10 except forL.sericata and C.rufifacies when 10 mM Tris-HCl , pH 8.0 was used.

Table 3 shows the inhibitory activity of the pooled 6 kD PI peptides(C1, T1, T2/T3, T4), the mixture of trypsin inhibitors T2/T3 and T4, andthe chymotrypsin inhibitor C1 against the proteases in the gut ofvarious members of the Lepidoptera, Coleoptera, Orthoptera and Diptera.In most cases, the pooled peptides and the trypsin inhibitors had anequivalent effect against the gut proteases with the degree ofinhibition ranging from 37-79% depending on the insect tested. Theinhibitors had negligible effect on the gut proteases of the potatotuber moth, P.opercullela. The chymotrypsin inhibitor C1 also affectedthe activity of the proteases but was less effective than the trypsininhibitors in five cases (W.cervinata, L.serricata, C.zealandica,P.octo, sugar cane grub).

The experimental details are described in the legend to FIG. 14. The N.alata PI was more effective than Soybean Bowman-Birk inhibitor inreducing cricket weight. It has shown that there is a good correlationbetween the ability of a proteinase inhibitor to inhibit the enzymes ofthe insect midgut and its effectiveness in retarding the growth ofinsects in insect feeding trials (Christeller et al., 1992). FIG. 14shows that the pooled PIs that inhibited the gut proteases of the blackfield cricket (T.commodus) by 70% in the in vitro assay retarded thegrowth of the crickets by 30% in a feeding trial conducted over a 10week period. The correlation between in vitro assays and feeding trialshas been confirmed recently by Johnston and collegues (1993) working ongrowth and development of Helicoverpa armigera.

                  TABLE 2                                                         ______________________________________                                        HPLC     retention time                                                                            molecular                                                  peak (min) weight assigned peptide*                                         ______________________________________                                        1        15.5        5731.5    C1                                                 5644.4 C1 minus Ser.sub.53                                                    5616.4 C1 minus Asp.sub.1 &                                                    Ser.sub.53                                                                   55.29.3 C1 minus Asp.sub.1                                                  2 20.5 5700.5 T2/T3                                                             5728.5 T4                                                                     5585.4 T2/T3 minus Asp.sub.1                                                  5613.5 T4 minus Asp.sub.1                                                   3 22.5 5725.5 T1                                                                5610.5 T1 minus Asp.sub.1                                                   4 24 5654.4 T1 minus Ala.sub.53                                                 5641.4 T4 minus Ser.sub.53                                                    5613.4 T2/T3 minus Ser.sub.53                                                 5539.4 T1 minus Asp.sub.1 &                                                    Ala.sub.53                                                                   5498.4 T2/T3 minus Asp.sub.1 &                                                 Ser.sub.53                                                                   5526.4 T4 minus Asp.sub.1 &                                                    Ser.sub.53                                                               ______________________________________                                         *See FIG. 9 for designation of C1 and T1-T4.                             

                  TABLE 3                                                         ______________________________________                                        Effect of Nicotiana alata proteinase inhibitors and Potato                      inhibitor II on casein hydrolysis by crude gut extracts                                casein hydrolysis (% control)                                                                     T2/T3,                                           Insect NaPI C1 T4                                                           ______________________________________                                        H. armigera                                                                              33.2         32.7   30.3                                             H. punctigera 26.6 29.3 28.5                                                  T. commodus 28.4 35.0 33.1                                                    A. infusa 37.5 40.2 43.3                                                      sugar cane 25.8 43.9 25.1                                                     grub                                                                          W. cervinata 22.9 82.9 20.4                                                   E. postvitiana 39.7 45.4 41.2                                                 S. litura 28.1 33.6 24.8                                                      P. opercullela 95.8 100 98.5                                                  C. rufifacies 29.1 37.8 28.9                                                  L. serricata 59.2 100 63.0                                                    C. zealandica 31.7 54.7 32.0                                                  P. octo 57.1 67.2 57.4                                                        C. obliquana 51.1 49.1 45.5                                                   A. tsmaniae 28.3 34.2 39.5                                                  ______________________________________                                         Legend to Table 3                                                             NaPI = N. alata proteinase inhibitors pooled                                  C1 = N. alata chymotrypsin inhibitor (peak 1 from HPLC)                       T2/T3, T4 = N. alata trypsin inhibitors (peak 2 from HPLC)                    Heliothis armigera, Helicoverpa armigera, Tobacco budworm, Lepidoptera        Heliothis punctigera, Helicoverpa punctigera Native budworm, Lepidoptera      Teleogryllus commodus Black field cricket, Orthoptera                         Agrotis infusa Common cutworm, adults known as the Bogong moth,               Lepidoptera                                                                   Wiseana cervinata Porina, native to New Zealand, Lepidoptera                  Lucilla sericata Green blow fly, Diptera, assayed at pH 8                     Chrysomya rufifacies Hairy maggot blow fly, Diptera, assayed at pH 8          Aphodius tasmaniae Tasmanian grass grub = Blackheaded pasture cockchafer,     Coleoptera                                                                    Costelytra zealandica New Zealand grass grub, Coleoptera                      Spodoptera litura Tropical armyworm, Lepidoptera                              Phthorimaea opercullela Potato tuber moth, Lepidoptera                        Epiphyas postvittana Lightbrown apple moth (leafroller), Lepidoptera          Planototrix octo Greenheaded leafroller, Lepidoptera                          Ctenopseustis obliquana Brownheaded leafroller, Lepidoptera                   Sugar cane grub                                                          

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

REFERENCES

Anderson, M. A., McFadden, G. I., Bernatzky, R., Atkinson, A., Orpin,T., Dedman, H., Tregear, G., Fernley, R., Clarke, A. E. (1989) The PlantCell 1:483-491.

Atkinson, A. H. (1992) PhD thesis, University of Melbourne, Victoria,Australia.

Bernatzky, R., Tanksley, S. D. (1986) Theor. Appl. Genet. 72:314-321.

Bradford, M. M. (1976) Anal. Biochem. 72:248-254

Brown, W. E., Ryan, C. A. (1984) Biochemistry 23:3418-3422.

Bryant, J., Green, T. R., Gurusaddaiah, T., Ryan, C. A. (1976)Biochemistry 15:3418-3424.

Cornish, E. C., Pettitt, J. M., Bonig, I., Clarke, A. E. (1987) Nature326: 99-102.

Christeller, J. T., Shaw, B. D., Gardiner, S. E., Dymock, J. (1989)Insect Biochem. 19: 2217-231.

Christeller, J. T., Laing, W. A., Markwick, N. P. and Burgess, E. P. J.(1992) Insect Biochem. Molec. Biol. 22:735-746

Feinberg, A. P., Vogelstein, B. (1983) Anal. Biochem. 132: 6-13.

Graham, J. S., Pearce, G., Merryweather, J., Titani, K., Ericsson, L.H., Ryan, C. A. (1985) J. Biol. Chem. 260: 6561-6564.

Graham, J. S., Hall, G., Pearce, G., Ryan, C. A. (1986) Planta169:399-405.

Grego, B., van Driel, I. R., Stearne, P. A., Goding, J. W., Nice, E. G.,Simpson, R. J. (1985) Eur. J. Biochem. 148:485-491.

Green, T. R., Ryan, C. A. (1972) Science: 776-777.

Hara-Nishimura, I., Inoue, K., Nishimura, M. (1991) FEBS Letters 29489-93.

Harlow, E., Lane, D. (1988) Antibodies. A Laboratory Manual. Cold SpringHarbour Laboratory, New York.

Hass, G. M., Hermodson, M. A., Ryan, C. A., Gentry, L. (1982)Biochemistry 21:752-756.

Johnston, K. A., Gatehouse, J. A., Anstee, J. H. (1993) J. InsectPhysiol. 39, 657-664.

King, L. A. Possee, R. D. (1992). The Baculovirus Expression system. ALaboratory guide. (Chapman & Hall: London, UK).

Kuo, J., Pearce, G. Ryan C. A. (1984) Isolation and characterization ofproteinase inhibitor I from etiolated tobacco leaves. Arch. Biochem.Biophys. 230: 504-510.

Kyte, J., Doolittle, R. F. (1982) J. Mol. Biol. 157: 680-685.

Laemmli, U. K. (1970) Nature 227:680-685.

Lucknow, V. A. and Summers, M. D. (1989). Virology. 170:31-39.

Mau, S-L., Williams, E. G., Atkinson, A., Anderson, M. A., Cornish, E.C., Grego, B., Simpson, R. J., Kheyr-Pour, A., Clarke, A. E. (1986)Planta 169:184-191.

Melville, J. C., Ryan, C. A. (1970) Archives of Biochemistry andBiophysics 138: 700-702.

Pearce, G., Ryan, C. A., Liljegren, D. (1988) Planta 175:527-531.

Plunkett, G., Senear, D. F., Zuroske, G., Ryan, C. A. (1982) Arch.Biochem. Biophys. 213: 463-472.

Richardson, M. (1977) Phytochemistry 16:159-169.

Rickauer, M., Fournier, J., Esquerre-Tugaye, M. (1989) Plant Physiol.9:1065-1070.

Ryan, C. A. (1984). Defense responses in plants. In: Plant GeneResearch, Dennes, E. S., Hohn, B., Hohn, T., King, P., Schell, J.,Verma, D. P. S., Eds. New York, Springer-Verlag, 375-386.

Sanchez-Serrano, J. J., Schmidt, R., Schell, J., Willmitzer, L., (1986)Mol. Gen. Genet. 203:15-20.

Scott, M. P., Jung, R., Muntz, K., Nielsen, N. C. (1992) Proc. Natl.Acad. Sci. USA 89 658-662.

Thornburg, R. W. An, G., Cleveland, T. E., Johnson, R., Ryan, C. A.(1987). Proc. Nat. Acad. Sci. USA 84:744-748.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 14                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1104 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - AAGGCTTGTA CCTTAAACTG TGATCCAAGA ATTGCCTATG GAGTTTGCCC GC -            #GTTCAGAA     60                                                                 - - GAAAAGAAGA ATGATCGGAT ATGCACCAAC TGTTGCGCAG GCACGAAGGG TT -            #GTAAGTAC    120                                                                 - - TTCAGTGATG ATGGAACTTT TGTTTGTGAA GGAGAGTCTG ATCCTAGAAA TC -            #CAAAGGCT    180                                                                 - - TGTACCTTAA ACTGTGATCC AAGAATTGCC TATGGAGTTT GCCCGCGTTC AG -            #AAGAAAAG    240                                                                 - - AAGAATGATC GGATATGCAC CAACTGTTGC GCAGGCACGA AGGGTTGTAA GT -            #ACTTCAGT    300                                                                 - - GATGATGGAA CTTTTGTTTG TGAAGGAGAG TCTGATCCTA GAAATCCAAA GG -            #CTTGTCCT    360                                                                 - - CGGAATTGCG ATCCAAGAAT TGCCTATGGG ATTTGCCCAC TTGCAGAAGA AA -            #AGAAGAAT    420                                                                 - - GATCGGATAT GCACCAACTG TTGCGCAGGC AAAAAGGGTT GTAAGTACTT TA -            #GTGATGAT    480                                                                 - - GGAACTTTTG TTTGTGAAGG AGAGTCTGAT CCTAAAAATC CAAAGGCCTG TC -            #CTCGGAAT    540                                                                 - - TGTGATGGAA GAATTGCCTA TGGGATTTGC CCACTTTCAG AAGAAAAGAA GA -            #ATGATCGG    600                                                                 - - ATATGCACCA ACTGCTGCGC AGGCAAAAAG GGTTGTAAGT ACTTTAGTGA TG -            #ATGGAACT    660                                                                 - - TTTGTTTGTG AAGGAGAGTC TGATCCTAAA AATCCAAAGG CTTGTCCTCG GA -            #ATTGTGAT    720                                                                 - - GGAAGAATTG CCTATGGGAT TTGCCCACTT TCAGAAGAAA AGAAGAATGA TC -            #GGATATGC    780                                                                 - - ACAAACTGTT GCGCAGGCAA AAAGGGCTGT AAGTACTTTA GTGATGATGG AA -            #CTTTTGTT    840                                                                 - - TGTGAAGGAG AGTCTGATCC TAGAAATCCA AAGGCCTGTC CTCGGAATTG TG -            #ATGGAAGA    900                                                                 - - ATTGCCTATG GAATTTGCCC ACTTTCAGAA GAAAAGAAGA ATGATCGGAT AT -            #GCACCAAT    960                                                                 - - TGTTGCGCAG GCAAGAAGGG CTGTAAGTAC TTTAGTGATG ATGGAACTTT TA -            #TTTGTGAA   1020                                                                 - - GGAGAATCTG AATATGCCAG CAAAGTGGAT GAATATGTTG GTGAAGTGGA GA -            #ATGATCTC   1080                                                                 - - CAGAAGTCTA AGGTTGCTGT TTCC          - #                  - #                  1104                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1360 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 97..1200                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - CGAGTAAGTA TGGCTGTTCA CAGAGTTAGT TTCCTTGCTC TCCTCCTCTT AT -             #TTGGAATG     60                                                                 - - TCTCTGCTTG TAAGCAATGT GGAACATGCA GATGCC AAG GCT TGT - #ACC TTA AAC          114                                                                                         - #                  - #    Lys Ala Cys Thr Leu Asn                           - #                  - #      1            - #   5           - - TGT GAT CCA AGA ATT GCC TAT GGA GTT TGC CC - #G CGT TCA GAA GAA AAG          162                                                                       Cys Asp Pro Arg Ile Ala Tyr Gly Val Cys Pr - #o Arg Ser Glu Glu Lys                        10     - #             15     - #             20                  - - AAG AAT GAT CGG ATA TGC ACC AAC TGT TGC GC - #A GGC ACG AAG GGT TGT          210                                                                       Lys Asn Asp Arg Ile Cys Thr Asn Cys Cys Al - #a Gly Thr Lys Gly Cys                    25         - #         30         - #         35                      - - AAG TAC TTC AGT GAT GAT GGA ACT TTT GTT TG - #T GAA GGA GAG TCT GAT          258                                                                       Lys Tyr Phe Ser Asp Asp Gly Thr Phe Val Cy - #s Glu Gly Glu Ser Asp                40             - #     45             - #     50                          - - CCT AGA AAT CCA AAG GCT TGT ACC TTA AAC TG - #T GAT CCA AGA ATT GCC          306                                                                       Pro Arg Asn Pro Lys Ala Cys Thr Leu Asn Cy - #s Asp Pro Arg Ile Ala            55                 - # 60                 - # 65                 - # 70       - - TAT GGA GTT TGC CCG CGT TCA GAA GAA AAG AA - #G AAT GAT CGG ATA TGC          354                                                                       Tyr Gly Val Cys Pro Arg Ser Glu Glu Lys Ly - #s Asn Asp Arg Ile Cys                            75 - #                 80 - #                 85              - - ACC AAC TGT TGC GCA GGC ACG AAG GGT TGT AA - #G TAC TTC AGT GAT GAT          402                                                                       Thr Asn Cys Cys Ala Gly Thr Lys Gly Cys Ly - #s Tyr Phe Ser Asp Asp                        90     - #             95     - #            100                  - - GGA ACT TTT GTT TGT GAA GGA GAG TCT GAT CC - #T AGA AAT CCA AAG GCT          450                                                                       Gly Thr Phe Val Cys Glu Gly Glu Ser Asp Pr - #o Arg Asn Pro Lys Ala                   105          - #       110          - #       115                      - - TGT CCT CGG AAT TGC GAT CCA AGA ATT GCC TA - #T GGG ATT TGC CCA CTT          498                                                                       Cys Pro Arg Asn Cys Asp Pro Arg Ile Ala Ty - #r Gly Ile Cys Pro Leu               120              - #   125              - #   130                          - - GCA GAA GAA AAG AAG AAT GAT CGG ATA TGC AC - #C AAC TGT TGC GCA GGC          546                                                                       Ala Glu Glu Lys Lys Asn Asp Arg Ile Cys Th - #r Asn Cys Cys Ala Gly           135                 1 - #40                 1 - #45                 1 -      #50                                                                              - - AAA AAG GGT TGT AAG TAC TTT AGT GAT GAT GG - #A ACT TTT GTT TGT        GAA      594                                                                    Lys Lys Gly Cys Lys Tyr Phe Ser Asp Asp Gl - #y Thr Phe Val Cys Glu                          155  - #               160  - #               165              - - GGA GAG TCT GAT CCT AAA AAT CCA AAG GCC TG - #T CCT CGG AAT TGT GAT          642                                                                       Gly Glu Ser Asp Pro Lys Asn Pro Lys Ala Cy - #s Pro Arg Asn Cys Asp                       170      - #           175      - #           180                  - - GGA AGA ATT GCC TAT GGG ATT TGC CCA CTT TC - #A GAA GAA AAG AAG AAT          690                                                                       Gly Arg Ile Ala Tyr Gly Ile Cys Pro Leu Se - #r Glu Glu Lys Lys Asn                   185          - #       190          - #       195                      - - GAT CGG ATA TGC ACC AAC TGC TGC GCA GGC AA - #A AAG GGT TGT AAG TAC          738                                                                       Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Ly - #s Lys Gly Cys Lys Tyr               200              - #   205              - #   210                          - - TTT AGT GAT GAT GGA ACT TTT GTT TGT GAA GG - #A GAG TCT GAT CCT AAA          786                                                                       Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gl - #y Glu Ser Asp Pro Lys           215                 2 - #20                 2 - #25                 2 -      #30                                                                              - - AAT CCA AAG GCT TGT CCT CGG AAT TGT GAT GG - #A AGA ATT GCC TAT        GGG      834                                                                    Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp Gl - #y Arg Ile Ala Tyr Gly                          235  - #               240  - #               245              - - ATT TGC CCA CTT TCA GAA GAA AAG AAG AAT GA - #T CGG ATA TGC ACA AAC          882                                                                       Ile Cys Pro Leu Ser Glu Glu Lys Lys Asn As - #p Arg Ile Cys Thr Asn                       250      - #           255      - #           260                  - - TGT TGC GCA GGC AAA AAG GGC TGT AAG TAC TT - #T AGT GAT GAT GGA ACT          930                                                                       Cys Cys Ala Gly Lys Lys Gly Cys Lys Tyr Ph - #e Ser Asp Asp Gly Thr                   265          - #       270          - #       275                      - - TTT GTT TGT GAA GGA GAG TCT GAT CCT AGA AA - #T CCA AAG GCC TGT CCT          978                                                                       Phe Val Cys Glu Gly Glu Ser Asp Pro Arg As - #n Pro Lys Ala Cys Pro               280              - #   285              - #   290                          - - CGG AAT TGT GAT GGA AGA ATT GCC TAT GGA AT - #T TGC CCA CTT TCA GAA         1026                                                                       Arg Asn Cys Asp Gly Arg Ile Ala Tyr Gly Il - #e Cys Pro Leu Ser Glu           295                 3 - #00                 3 - #05                 3 -      #10                                                                              - - GAA AAG AAG AAT GAT CGG ATA TGC ACC AAT TG - #T TGC GCA GGC AAG        AAG     1074                                                                    Glu Lys Lys Asn Asp Arg Ile Cys Thr Asn Cy - #s Cys Ala Gly Lys Lys                          315  - #               320  - #               325              - - GGC TGT AAG TAC TTT AGT GAT GAT GGA ACT TT - #T ATT TGT GAA GGA GAA         1122                                                                       Gly Cys Lys Tyr Phe Ser Asp Asp Gly Thr Ph - #e Ile Cys Glu Gly Glu                       330      - #           335      - #           340                  - - TCT GAA TAT GCC AGC AAA GTG GAT GAA TAT GT - #T GGT GAA GTG GAG AAT         1170                                                                       Ser Glu Tyr Ala Ser Lys Val Asp Glu Tyr Va - #l Gly Glu Val Glu Asn                   345          - #       350          - #       355                      - - GAT CTC CAG AAG TCT AAG GTT GCT GTT TCC TA - #AGTCCTAA CTAATAATAT           1220                                                                       Asp Leu Gln Lys Ser Lys Val Ala Val Ser                                           360              - #   365                                                 - - GTAGTCTATG TATGAAACAA AGGCATGCCA ATATGCTCTG TCTTGCCTGT AA -             #TCTGTAAT   1280                                                                 - - ATGGTAGTGG AGCTTTTCCA CTGCCTGTTT AATAAGAAAT GGAGCACTAG TT -            #TGTTTTAG   1340                                                                 - - TTAAAAAAAA AAAAAAAAAA            - #                  - #                     136 - #0                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 368 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Lys Ala Cys Thr Leu Asn Cys Asp Pro Arg Il - #e Ala Tyr Gly Val Cys        1               5 - #                 10 - #                 15              - - Pro Arg Ser Glu Glu Lys Lys Asn Asp Arg Il - #e Cys Thr Asn Cys Cys                   20     - #             25     - #             30                  - - Ala Gly Thr Lys Gly Cys Lys Tyr Phe Ser As - #p Asp Gly Thr Phe Val               35         - #         40         - #         45                      - - Cys Glu Gly Glu Ser Asp Pro Arg Asn Pro Ly - #s Ala Cys Thr Leu Asn           50             - #     55             - #     60                          - - Cys Asp Pro Arg Ile Ala Tyr Gly Val Cys Pr - #o Arg Ser Glu Glu Lys       65                 - # 70                 - # 75                 - # 80       - - Lys Asn Asp Arg Ile Cys Thr Asn Cys Cys Al - #a Gly Thr Lys Gly Cys                       85 - #                 90 - #                 95              - - Lys Tyr Phe Ser Asp Asp Gly Thr Phe Val Cy - #s Glu Gly Glu Ser Asp                  100      - #           105      - #           110                  - - Pro Arg Asn Pro Lys Ala Cys Pro Arg Asn Cy - #s Asp Pro Arg Ile Ala              115          - #       120          - #       125                      - - Tyr Gly Ile Cys Pro Leu Ala Glu Glu Lys Ly - #s Asn Asp Arg Ile Cys          130              - #   135              - #   140                          - - Thr Asn Cys Cys Ala Gly Lys Lys Gly Cys Ly - #s Tyr Phe Ser Asp Asp      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gly Thr Phe Val Cys Glu Gly Glu Ser Asp Pr - #o Lys Asn Pro Lys        Ala                                                                                             165  - #               170  - #               175             - - Cys Pro Arg Asn Cys Asp Gly Arg Ile Ala Ty - #r Gly Ile Cys Pro Leu                  180      - #           185      - #           190                  - - Ser Glu Glu Lys Lys Asn Asp Arg Ile Cys Th - #r Asn Cys Cys Ala Gly              195          - #       200          - #       205                      - - Lys Lys Gly Cys Lys Tyr Phe Ser Asp Asp Gl - #y Thr Phe Val Cys Glu          210              - #   215              - #   220                          - - Gly Glu Ser Asp Pro Lys Asn Pro Lys Ala Cy - #s Pro Arg Asn Cys Asp      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Gly Arg Ile Ala Tyr Gly Ile Cys Pro Leu Se - #r Glu Glu Lys Lys        Asn                                                                                             245  - #               250  - #               255             - - Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Ly - #s Lys Gly Cys Lys Tyr                  260      - #           265      - #           270                  - - Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gl - #y Glu Ser Asp Pro Arg              275          - #       280          - #       285                      - - Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp Gl - #y Arg Ile Ala Tyr Gly          290              - #   295              - #   300                          - - Ile Cys Pro Leu Ser Glu Glu Lys Lys Asn As - #p Arg Ile Cys Thr Asn      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Cys Cys Ala Gly Lys Lys Gly Cys Lys Tyr Ph - #e Ser Asp Asp Gly        Thr                                                                                             325  - #               330  - #               335             - - Phe Ile Cys Glu Gly Glu Ser Glu Tyr Ala Se - #r Lys Val Asp Glu Tyr                  340      - #           345      - #           350                  - - Val Gly Glu Val Glu Asn Asp Leu Gln Lys Se - #r Lys Val Ala Val Ser              355          - #       360          - #       365                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Lys Ala Cys Thr Leu Asn Cys Asp Pro Arg Il - #e Ala Tyr Gly Val Cys      1               5   - #                10  - #                15               - - Pro Arg Ser Glu Glu Lys Lys Asn                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Th - #r Lys Gly Cys Lys Tyr      1               5   - #                10  - #                15               - - Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gl - #y Glu Ser Asp Pro Arg                  20      - #            25      - #            30                   - - Asn Pro Lys Ala Cys Thr Leu Asn Cys Asp Pr - #o Arg Ile Ala Tyr Gly              35          - #        40          - #        45                       - - Val Cys Pro Arg Ser Glu Glu Lys Lys Asn                                      50              - #    55                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Th - #r Lys Gly Cys Lys Tyr      1               5   - #                10  - #                15               - - Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gl - #y Glu Ser Asp Pro Arg                  20      - #            25      - #            30                   - - Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp Pr - #o Arg Ile Ala Tyr Gly              35          - #        40          - #        45                       - - Ile Cys Pro Leu Ala Glu Glu Lys Lys Asn                                      50              - #    55                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Ly - #s Lys Gly Cys Lys Tyr      1               5   - #                10  - #                15               - - Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gl - #y Glu Ser Asp Pro Lys                  20      - #            25      - #            30                   - - Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp Gl - #y Arg Ile Ala Tyr Gly              35          - #        40          - #        45                       - - Ile Cys Pro Leu Ser Glu Glu Lys Lys Asn                                      50              - #    55                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Ly - #s Lys Gly Cys Lys Tyr      1               5   - #                10  - #                15               - - Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gl - #y Glu Ser Asp Pro Lys                  20      - #            25      - #            30                   - - Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp Gl - #y Arg Ile Ala Tyr Gly              35          - #        40          - #        45                       - - Ile Cys Pro Leu Ser Glu Glu Lys Lys Asn                                      50              - #    55                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Ly - #s Lys Gly Cys Lys Tyr      1               5   - #                10  - #                15               - - Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gl - #y Glu Ser Asp Pro Arg                  20      - #            25      - #            30                   - - Asn Pro Lys Ala Cys Pro Arg Asn Cys Pro Gl - #y Arg Ile Ala Tyr Gly              35          - #        40          - #        45                       - - Ile Cys Pro Leu Ser Glu Glu Lys Lys Asn                                      50              - #    55                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Ly - #s Lys Gly Cys Lys Tyr      1               5   - #                10  - #                15               - - Phe Ser Asp Asp Gly Thr Phe Ile Cys Glu Gl - #y Glu Ser Glu Thr Ala                  20      - #            25      - #            30                   - - Ser Lys Val Asp Glu Tyr Val Gly Glu Val Gl - #u Asn Asp Leu Gln Lys              35          - #        40          - #        45                       - - Ser Lys Val Ala Val Ser                                                      50                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Th - #r Lys Gly                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Ly - #s Lys Gly                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Lys Ala Cys Thr Leu Asn                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Glu Glu Lys Lys Asn                                                      1               5                                                            __________________________________________________________________________

We claim:
 1. An isolated nucleic acid comprising a sequence ofnucleotides which encodes or is complementary to a sequence whichencodes a type II serine proteinase inhibitor (PI) precursor from aplant wherein said isolated nucleic acid has the nucleotide secuence setforth in SEQ ID NO:1 or hybridizes to the nucleotide sequence set forthin SEQ ID NO:1 under the conditions of at least one of 4×SSC at roomtemperature, 2×SSC at room temperature, 1×SSC at 40° C., 2×SSC with 0.1%w/v SDS at 68° C., or 0.2×SSC with 1% w/v SDS at 68° C., wherein saidprecursor comprises at least three PI monomers and wherein at least oneof said monomers has a chymotrypsin specific site and at least one ofsaid monomers has a trypsin specific site.
 2. An isolated nucleic acidaccording to claim 1 wherein said PI precursor comprises at least fourmonomers.
 3. An isolated nucleic acid according to claim 1 wherein thePI precursor comprises at least five monomers.
 4. An isolated nucleicacid according to claim 1 wherein the PI precursor comprises at leastsix monomers.
 5. An isolated nucleic acid comprising a sequence ofnucleotides according to claim 1 which encodes or is complementary to asequence which encodes a single type II serine proteinase inhibitor (PI)having either a chymotrypsin specific site or a trypsin specific siteand wherein said PI is a monomer of a precursor PI having at least threemonomers of which at least one of said monomers has a chymotrypsin siteand the other of said monomers has a trypsin site.
 6. An isolatednucleic acid according to claim 1 or claim 5 which encodes a peptideselected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO:9, or SEQ ID NO:10.
 7. Amethod of increasing or enhancing resistance of a plant to insect orother pathogen infestation, said method comprising introducing a nucleicacid molecule as defined in any one of claims 1, 2, 3, 4, or 5 into acell or group of cells of said plant, regenerating a plant therefrom andgrowing said plant for a time and under conditions sufficient to permitexpression of said nucleic acid into a proteinase inhibitor (PI) orprecursor thereof which inhibits growth and infestation by saidpathogen.